Abstract Hox genes, especially HOXA and HOXB genes, are critical for maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Dysregulation of HOXA and/or HOXB genes is a dominant mechanism of leukemic transformation. Aberrant HOX gene expression is associated with fusion genes involving MLL1 and NUP98, and mutations in NPM1 and CEBPA. However, the epigenetic mechanisms that regulate HOX gene transcription to control HSC function remain to be explored. Furthermore, it is critical to elucidate how HOX genes are aberrantly activated during leukemogenesis. Better understanding of these critical questions will assist in the development of highly effective and selective targeted therapies. We recently identified a HoxB locus associated long intergenic noncoding RNA (lncRNA), HoxBlinc, which controls hematopoietic lineage commitment and differentiation by organizing CTCF mediated active chromatin domain to facilitate anterior HoxB gene activation. HoxBlinc recruits the Setd1a/MLL1 complexes to activate HoxB genes. We found that HoxBlinc lncRNA is overexpressed in significant portions of AML patients, and AML patients with high HoxBlinc expression had significantly shortened survival compare to patients with low HOCBLINC expression. Furthermore, transgenic expression of HoxBlinc in mice leads to increased pools of LT-HSCs and ST-HSCs, and development of lethal AML-like disease. We hypothesize that HoxBlinc lncRNA is a critical epigenetic regulator of HSCs, by controlling the activation of Hox and other key HSC-regulating genes through modulation of chromatin dynamics. In addition, up-regulation of HoxBlinc may represent a potent oncogenic event in leukemogenesis. To test these hypothesis, we will: 1) determine the role of HoxBlinc lncRNA in HSC biology and behavior by performing serial transplantation and paired daughter cell assays using purified HoxBlinc-Tg HSCs; 2) determine whether transgenic HoxBlinc expression is sufficient to perturb hematopoiesis and cause myeloid malignancies in mice; 3) investigate the mechanism(s) by which HoxBlinc lncRNA regulates behaviors of different stages of HSCs by examining global changes in HoxBlinc chromatin binding, 3D chromatin organization, histone modifications, chromatin accessibility, as well as transcription profiles in HSCs purified from WT and HoxBlinc-Tg mice; 4) examine if the function exerted by HoxBlinc lncRNA in HSCs is dependent on the Setd1a/MLL1 complexes; 5) explore whether HOXBLINC lncRNA can serve as an effective therapeutic target for AMLs by examining whether HoxBlinc loss is capable of mitigating NPM1C+- or other mutation-driven myeloid malignancies via abrogating the signature aberrant HOX gene expression. Success of the proposed studies will result in fundamental knowledge regarding the regulation of HSCs by lncRNAs. Our studies could establish HoxBlinc as a powerful oncogenic lncRNAs during leukemogenesis. HOXBLINC lncRNA might represent a novel therapeutic target for AMLs with high HOXBLINC expression.